Collagen scaffolds, medical implants with same and methods of use

ABSTRACT

The subject invention concerns non-degradable three dimensional porous collagen scaffolds and coatings. These scaffolds can be prepared around sensors for implantation into a body. A specific embodiment of the invention concerns implantable glucose sensors. Sensors comprising a collagen scaffold of the invention have improved biocompatibility by minimizing tissue reactions while stimulating angiogenesis. The subject invention also concerns methods for preparing collagen scaffolds of the invention. The subject invention also concerns sensors that have a collagen scaffold of the invention around the exterior of the sensor.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/805,495, filed Jun. 22, 2006, which is herebyincorporated by reference herein in its entirety, including any figures,tables, nucleic acid sequences, amino acid sequences, and drawings.

GOVERNMENT SUPPORT

This invention was made with Government support under NationalInstitutes of Health Grant No. 1R01 EB001640-01. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

This invention relates to implantable biosensors and medical devices.More specifically, this invention relates to collagen scaffold coveringsfor implantable sensors and/or other medical devices promotingbiocompatibility.

BACKGROUND OF THE INVENTION

Chronically implantable devices may provoke inflammation and/or fibrosisfrom tissue trauma or tissue response to the foreign body. See, e.g.,Reichert et al., Handbook of Biomaterial Evaluation, Ch. 28 Biosensors,pp. 439-460, (Von Recum A., editor) (1999); Wisniewski et al., J AnalChem 2000; 366 (6-7) (p. 611-621).

Implanted devices may also cause other unwanted bioreactions. Forexample, recently, researchers have stated that drug-coated stents mightcause adverse reactions leading to blood clot formation in somepatients. See Lagerqvist et al., Long-Term Outcomes with Drug-ElutingStents versus Bare-Metal Stents in Sweden, New England Jnl. Of Medicine,Mar. 8, 2007.

Other chronically implantable devices that may invoke unwantedbioreactions are biosensors. For example, in order to maintain nearnormal blood glucose levels (70-120 mg/dL), diabetic patients widely useover-the-counter glucose meters, which require finger pricking to obtainblood samples several times a day. The pain (Lee et al., 2005),inconvenience, and discomfort of self-monitoring of blood glucose (SMBG)are frequently obstacles to effective patient compliance and optimalmanagement of diabetes. During the past 20 years many kinds ofcontinuous glucose monitoring systems have been studied includingsensors implanted in the subcutaneous tissue (Moussy et al., 1993;Johnson et al., 1992; Koudelka et al., 1991; Bindra et al., 1991; Pickupet al., 1989; Shichiri et al., 1986; and Ertefai et al, 1989), sensorsimplanted in the vascular bed (Armour et al., 1990; Frost et al., 2002),and determining glucose concentration in interstitial fluid sampledusing a micro dialysis device (Ash et al., 1992; Meyerhoff et al., 1992;Moscone et al., 1992). Although several studies of implantable glucosesensors have been reported, it is believed that none of these biosensorsare reliably capable of continuously monitoring glucose levels duringlong-term implantation. Progressive loss of sensor function occurs duein part to biofouling and to the consequences of a foreign body responsesuch as inflammation, fibrosis, and loss of vasculature (Reichert etal., 1992; Reichert et al., 1999; Sharkawy et al., 2007). Someresearchers have modified the surface of the sensors to reduce membranebiofouling in vivo. In an approach to reduce protein adsorption, Quinnet al., 1995 used poly(ethylene glycol) (PEG) in apolyhydroxyethylmethacrylate (PHEMA) matrix. Since the PEG chains tendto stand up perpendicular to the membrane surface, they provide awater-rich phase that resists binding of many protein molecules. Rigbyet al., 1995 and Reddy et al., 1997 reduced protein adsorption by usingdiamond-like carbon, so-called “inert” materials. Shichiri et al., 1988incorporated an alginate/polylysine gel layer at the sensor. Shaw etal., 1991 reported improvement in biocompatibility of a biosensor coatedwith PHEMA/PU (polyurethane). Wilkins et al., 1995 and Moussy et al.introduced NAFION (perfluorosulphonic acid) membrane (Du Pont), toreduce “biofouling” on the surface of the sensor and reduce interferencefrom urate and ascorbate (Moussy et al., 1993; Moussy et al., 1994a;Moussy et al., 1994b; Moussy et al., 1994c). Armour et al., 1990 coatedtheir sensor tips with cross-linked albumin and Kemer et al., 1993developed cellulose-coated sensors to improve sensor bloodcompatibility. However, it is believed that none of these approaches hasbeen satisfactory for long term, stable glucose monitoring.

Collagen and its derived matrices are used extensively as naturalpolymers in the biomedical field including tissue engineering due to itslow antigenicity, its biodegradability and its good mechanical,haemostatic and cell-binding properties (Sheu et al., 2001; Pieper etal., 2002; Chvapil et al., 1973; Pachence et al., 1996; and Lee et al.,2001). In order to devise strategies for using collagen in thedevelopment of advanced biomaterials for biomedical engineering, it istypically desired to confer mechanical strength and resistance toenzymatic (collagenase) degradation resistance with chemical or physicalcross-linking strategies. There are several strategies for cross-linkingcollagen-based biomaterials. Glutaraldehyde (GA) is the most widely usedas a cross-linking agent for collagen-based biomaterials (Sheu et al.,2001; Barbani et al., 1995). However, GA and its reaction products areassociated with cytotoxicity in vivo, due to the presence ofcross-linking byproducts and the release of GA-linked collagen peptidesduring enzymatic degradation (Huang-Lee et al., 1990; van Luyn et al.,1992).

In order to avoid in vivo cytotoxicity and subsequent calcification ofGA cross-linked collagen, several alternative compounds have beenexamined as potential collagen cross-linking agents (Khor et al., 1997;Sung et al., 1996) such as polyepoxy, hexamethylene diisocyanate (HMDI),1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC), and ultra-violet(UV) or gamma-ray irradiation. Koob et al. recently described a processfor cross-linking of type I collagen fibers with nordihydroguaiareticacid (NDGA), a plant compound with antioxidant properties (Koob et al.,2002a; Koob et al., 2002b; Koob et al., 2001a; and Koob et al., 2001b).Koob et al. showed that NDGA significantly improved the mechanicalproperties of synthetic collagen fibers. In addition, they showed thatNDGA cross-linked collagen fibers did not elicit a foreign body responsenor did they stimulate an immune reaction during six weeks in vivo.

The extent of cross-linking and choice of cross-linking agent may alsoaffect the porosity and pore size of the scaffold and may influencefibrous capsule thickness, blood vessel density, and the location ofvessels within the three-dimensional porous scaffold (Joseph et al.,2004). Large pore scaffolds (greater than 60 micron pore size) allowdeep penetration of capillaries and supporting extracellular matrix(ECM). Sharkawy et al., 1997 showed that after four weeks ofsubcutaneous implantation in rat, a well-organized collagen matrixtypical of a foreign-body response encapsulated non-porous implants,while the porous polyvinyl alcohol (PVA) implants produced less fibrousand vascularized tissue capsules.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention provide a biocompatible collagen coveringfor medical devices to provide improved biocompatibility and/or reducedrisk of adverse reactions. Embodiments of the invention may beparticularly suitable as coverings and/or scaffolds for chronicallyimplantable medical devices.

Some embodiments are directed to non-degradable, biocompatible, threedimensional porous collagen scaffolds. These scaffolds can be formed orplaced on and/or prepared around devices and/or sensors for implantationinto a body. Some particular embodiments of the invention areimplantable glucose sensors with a porous collagen scaffold on anexternal surface thereof. Sensors comprising a collagen scaffold of theinvention can have improved biocompatibility by reducing tissuereactions while stimulating angiogenesis.

Other embodiments of the invention are directed to methods for preparingcollagen scaffolds. Three dimensional porous collagen scaffolds can befabricated by using a freeze-drying method and cross-linking them usingdifferent concentrations of at least one of glutaraldehyde (GA),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and/ornordihydroguaiaretic acid (NDGA) solution.

One embodiment of the present invention concerns a biocompatiblecollagen scaffold and/or coating for use with a device implantable inthe body or tissue of a person or animal, wherein said scaffold orcoating comprises a polymer intercalated into said scaffold or coating.

One embodiment of the present invention also concerns a method forpreparing a device for implantation into the body or tissue of a personor animal, said method comprising placing a biocompatible collagenscaffold or coating on said device wherein said scaffold or coatingcomprises a polymer intercalated into said scaffold or coating.

One embodiment of the present invention also concerns a method forproviding an implantable device with a biocompatible collagen coating orscaffold, said method comprising:

a) contacting an implantable device structure with a collagen containingsolution;

b) drying said collagen solution on said structure; and

c) embedding said collagen of said structure in a polymer matrix.

One embodiment of the present invention also concerns a device havingenhanced biocompatibility for implantation into the body or tissue of aperson or animal, wherein said device comprises a biocompatible collagenscaffold and/or coating comprising a polymer intercalated into saidscaffold or coating.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an exemplary scaffold-coated sensingelement of a glucose electrode according to embodiments of the presentinvention FIGS. 2A-2B are schematic illustrations of a chemicalmechanism for GA (FIG. 2A) and NDGA (FIG. 2B) cross-linking of acollagen scaffold according to embodiments of the present invention.

FIGS. 3A-3C are scanning electron micrographs (SEM) which show SEMmorphology of exemplary collagen scaffolds according to embodiments ofthe invention. Determination of the pore size of collagen scaffolds bySEM. FIG. 3A shows a collagen scaffold with no cross-linking (200×; 25.0kV); FIG. 3B shows a collagen scaffold with GA cross-linking (200×; 25.0kV); and FIG. 3C shows a collagen scaffold with NDGA cross-linking(200×; 25.0 kV).

FIG. 4 is a bar graph of degree (%) of cross-linking and degree (%) ofwater absorption of GA and NDGA cross-linked scaffolds that shows bulkproperties of GA and NDGA cross-linked scaffolds according toembodiments of the present invention. Results are shown as means ±SD(n=3).

FIG. 5 is a bar graph of % (original weight) verses collagenasetreatment time (weeks) that shows collagenase resistance of GA and NDGAcross-linked scaffolds in vitro. Results are shown as means ±SD (n=3).

FIGS. 6A-6D are SEMs that show SEM morphology of the scaffold after invitro degradation study. FIG. 6A shows NDGA cross-linked scaffold aftertwo weeks collagenase treatment (200×; 25.0 kV); FIG. 6B shows GAcross-linked scaffold after two weeks collagenase treatment (200×; 25.0kV); FIG. 6C shows NDGA cross-linked scaffold after four weekscollagenase treatment (200×; 25.0 kV); FIG. 6D shows GA cross-linkedscaffold after four weeks collagenase treatment (200×; 25.0 kV).

FIGS. 7A-7F are digital photographs that show in vivo stability of GAand NDGA cross-linked scaffolds in rat subcutaneous tissue. FIGS. 7A-7C:two weeks after implantation; FIG. 7A shows NDGA cross-linked scaffold;FIG. 7B shows GA cross-linked scaffold; FIG. 7C (left side) shows NDGAcross-linked scaffold; and FIG. 7C (right side) shows GA cross-linkedscaffold. FIGS. 7D-7F: four weeks after implantation; FIG. 7D shows NDGAcross-linked; FIG. 7E shows GA cross-linked scaffold; FIG. 7F (leftside) shows NDGA cross-linked scaffold; and FIG. 7F (right side) showsGA cross-linked scaffold.

FIGS. 8A-8D are light microscope digital pictures of the implantableglucose sensing element (FIG. 8A shows uncoated sensor; FIG. 8B showssensor coated with scaffold) and SEM morphology (FIG. 8C shows surface;FIG. 8D shows cross-section) of the scaffold region.

FIG. 9 is a graph of current (nA) versus time (minutes) that showsamperometric response curves of the glucose sensors (response curve1-uncoated sensor; response curve 2-coated with GA cross-linkedscaffold; and response curve 3-coated with NDGA cross-linked scaffold)from 5 mM to 15 mM glucose concentration. T_(95%), is defined as thetime at 95% of the maximum current change (I_(15 mM)−I_(5 mM)).

FIG. 10 is a graph of current (nA) versus glucose concentration (mM)that shows amperometric responses of uncoated and collagenscaffold-coated glucose sensors (2-30 mM glucose). The control (noscaffold) is shown by filled-in squares; GA cross-linked scaffold isshown by filled-in circles; NDGA cross-linked scaffold is shown byfilled-in triangles. Results are shown as means ±SD (n=3).

FIG. 11 is a graph of change in sensitivity (%) versus number of dippingcycles that shows the effect of the scaffold thickness on glucose sensorsensitivity. Results are shown as means ±SD (n=3).

FIGS. 12A-12L are digital images taken over time showing theinflammation response (Direct implantation-Histological Assay) ofbiosensors. FIGS. 12A-12F show GA cross-linked scaffolds: FIG. 12A (3days); FIG. 12B (7 days); FIG. 12C (14 days); FIG. 12D (21 days); FIG.12E (28 days); FIG. 12F (49 days). FIGS. 12G-12L show NDGA cross-linkedscaffolds: FIG. 12G (3 days) FIG. 12H (7 days); FIG. 12I (14 days); FIG.12J (21 days); FIG. 12K (28 days); FIG. 12L (49 days). There is lessinflammation associated with the NDGA cross-linked scaffold.

FIG. 13 shows an implantable glucose sensor. The double dotted linerepresents the skin and all components of glucose sensor shown to theleft of the double dotted line are implanted in the skin; all componentsof the glucose sensor shown to the right of the double dotted line areoutside the skin.

FIG. 14 shows implantable glucose sensors. The glucose sensor in theupper portion of the figure has a long wire (30 mm). The glucose sensorin the lower portion of the figure has a short wire (10 mm).

FIGS. 15A-15C show the in vivo study with the glucose sensors implantedin the backs of rats.

FIG. 16 shows the hydrated collagen scaffold. The left side is GAcross-linked collagen scaffold and the right side is the NDGA-reinforcedcollagen scaffold.

FIG. 17 is a graph of change in sensitivity (%) of the sensor versustime; the control (no scaffold) is shown by filled-in squares; thesensor with NDGA cross-linked scaffold is shown by filled in circles;the sensor with GA cross-linked scaffold is shown by filled-intriangles.

FIG. 18 is a graph of change in sensitivity (%) of sensor versusimplantation periods (weeks). The control (no scaffold) with ashort-wire sensor is shown by filled-in squares (6 working sensors outof 8 implanted sensors); the control (no scaffold) with a long-wiresensor is shown by open squares (4 working sensors out of 8 implantedsensors); the short-wire sensor with NDGA cross-linked scaffold is shownby filled-in circles (4 working sensors out of 8 implanted sensors); thelong-wire sensor with is NDGA cross-linked scaffold is shown by opencircles (2 working sensors out of 8 implanted sensors); the short-wiresensor with GA cross-linked scaffold is shown by filled-in triangles (4working sensors out of 8 implanted sensors); the long-wire sensor withGA cross-linked scaffold is shown by open triangles (1 working sensorout of 8 implanted sensors).

FIG. 19A-19C are photographs of the implanted glucose sensor showingphysical stability of the collagen scaffold after four weeks ofimplantation. FIG. 19A shows a detached implanted long-wire glucosesensor with NDGA reinforced collagen scaffold.

FIG. 19B shows a stable implanted short-wire glucose sensor with NDGAreinforced collagen scaffold. FIG. 19C shows an implanted short- and/orlong-wire glucose sensor with GA cross-linked collagen scaffold.

FIGS. 20A-20D are digital images of tissue subjected to histologicalassay (scaffold without sensors). FIG. 20A shows GA cross-linkedscaffolds (after two weeks implantation); FIG. 20B shows NDGAcross-linked scaffolds (after two weeks implantation); FIG. 20C shows GAcross-linked scaffolds (after four weeks implantation); FIG. 20B showsNDGA cross-linked scaffolds (after four weeks implantation).

FIG. 21 shows a flow chart for a method for preparing a device accordingto the present invention comprising a biocompatible collagen scaffold.Dashed lines indicate optional steps.

Further features, advantages and details of the present invention willbe appreciated by those of ordinary skill in the art from a reading ofthe figures and the detailed description of the embodiments that follow,such description being merely illustrative of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Generally stated, embodiments of the subject invention are directed tocollagen coverings, coatings and/or scaffolds which are particularlysuitable for implantable medical devices, and methods of making andusing the same in animal or human patients. The patient can be a humanor other animal, such as a primate, equine, bovine, ovine, canine, orfeline animal. The coatings, coverings and/or scaffolds can be providedas a tissue-contacting surface which may encapsulate all or a portion ofthe implantable devices to thereby provide a reduced immunogenicresponse and/or long-lived in vivo functionality of the implanteddevice.

The present invention now is described more fully hereinafter withreference to the accompanying drawings, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, thethickness of certain lines, layers, components, elements or features maybe exaggerated for clarity. Broken lines illustrate optional features oroperations unless specified otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. As used herein, phrases such as “between X and Y” and“between about X and Y” should be interpreted to include X and Y. Asused herein, phrases such as “between about X and Y” mean “between aboutX and about Y”. As used herein, phrases such as “from about X to Y” mean“from about X to about Y”.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being “on”,“attached” to, “connected” to, “coupled” with, “contacting’, etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on”, “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention. The sequence of operations (orsteps) is not limited to the order presented in the claims or figuresunless specifically indicated otherwise.

The term “implantable” means the device can be inserted, embedded,grafted or otherwise acutely or chronically attached or placed on or ina patient. The term “tissue” means skin, muscle, bone or other group ofcells. The term “chronically” means that the device is configured toremain implanted for at least 2 months, typically at least 6 months, andin some embodiments, one or more years while remaining operational forits intended function. The terms “coating” or “covering” refer to amaterial on a target surface of the device. The coating can be a porouscoating that can inhibit cell and tissue fouling of the underlyingdevice. The coating may not promote tissue growth. The coating can be athin or thick film, foam or other barrier to tissue fouling andbiodegradation. The term “scaffold” refers to a porous material and/orstructure into which cells, tissue, vessels, etc . . . , can grow into,colonize and populate. The scaffold can inhibit cell and tissue foulingof the device and/or reduce foreign body inflammatory and immunogenicresponses thereby prolonging the functional lifetime of the indwellingdevice.

Collagen “microfibrils,” “fibrils,” “fibers,” and “natural fibers” referto naturally-occurring structures found in a tendon. Microfibrils areabout 3.5 to 50 nm in diameter. Fibrils are about 50 nm to 50 μm indiameter. Natural fibers are above 50 μm in diameter. A “syntheticfiber” refers to any fiber-like material that has been formed and/orchemically or physically created or altered from its naturally-occurringstate. For example, an extruded fiber of fibrils formed from a digestedtendon is a synthetic fiber but a tendon fiber newly harvested from amammal is a natural fiber. Of course, synthetic collagen fibers caninclude non-collagenous components, such as hydroxyapatite or drugs thatfacilitate tissue growth. For example, the compositions can containgrowth factors such as basic fibroblast growth factor, tumor growthfactor beta, bone morphogenic proteins, platelet-derived growth factor,and insulin-like growth factors; chemotactic factors such fibronectinand hyaluronan; and extracellular matrix molecules such as aggrecan,biglycan, and decorin. Of course, synthetic collagen fibers can includenon-collagenous components, such as particulates, hydroxyapatite andother mineral phases, or drugs that facilitate tissue growth. Forexample, the compositions can contain carbon nano-tubes, zincnano-wires, nano-crystalline diamond, or other nano-scale particulates;larger crystalline and non-crystalline particulates such as calciumphosphate, calcium sulfate, apatite minerals. For example, thecompositions can contain therapeutic agents such as bisphosphonates,anti-inflammatory steroids, growth factors such as basic fibroblastgrowth factor, tumor growth factor beta, bone morphogenic proteins,platelet-derived growth factor, and insulin-like growth factors;chemotactic factors such fibronectin and hyaluronan; and extracellularmatrix molecules such as aggrecan, biglycan, and decorin.

Examples of devices that can benefit from the collagen coatings and/orscaffolds contemplated by embodiments of the invention, include, but arenot limited to, implantable stents, including cardiac, arterial, neuro(brain), urinary, and other stents, implantable power generators (IPGs),pacemakers, defibrillators, cardioverters, stimulators and/or leadsystems for the brain, central nervous system (CNS) or peripheralnervous system, cardiac or other biological system, cardiac replacementvalves, implantable sensors including glucose sensors, cardiac sensors,identity or tracking sensors (e.g., RFID), sensors to detect or measureO₂, pH, temperature, ions, and the like, orthopedic implants, includingtissue implants, such as facial implants for the chin, cheek, jawbone,and nose, implantable subcutaneous or percutaneous access ports, draintubes such as Eustachian drain tubes, catheters such as urinarycatheters, respiratory-assist tubes, and the like.

The collagen scaffold or covering of fibers can be configured tosubstantially encase the target implantable device or may cover only aportion thereof.

The scaffold or covering can be a three dimensional array of fibers orfibrils held together or on the device in any suitable manner includingby their natural affinity to stick together upon compression orextrusion, by using a sticky coating or adhesive, such as a gelatinouscoating, or by otherwise attaching the fibers to form the array. Thescaffold or coverings may also optionally comprise extruded,electrospun, braided and/or mesh collagen segments. The term “braided”and derivatives thereof mean to (inter)weave and/or interlock in anymanner, three or more fibers or bundles of fibers together, includingknitting and knotting and combinations of these or other interlockingconstructions. The collagen can be provided as laminate fibers, foams,electrospun yarns or other formations.

In some embodiments, the scaffold or covering can be configured as adrug delivery device for short term or long-term release or elution. Forexample, hydrogel matrices may be integrated into or on the collagenscaffold or covering.

In some embodiments, an NDGA treated collagen scaffold or collagencoating is applied to a biosensor or other implantable medical devices.The collagen scaffold or coating of the invention improvesbiocompatibility and longevity of sensors and devices implanted in abody or tissue. The use of collagen scaffold and/or coating ofembodiments of the present invention reduces the effect of tissuereactions (i.e., inflammation and fibrosis) on implanted biosensors andmedical devices.

In some embodiments, a non-degradable three-dimensional porous collagenscaffold is provided around an implantable glucose sensor tosubstantially encapsulate at least a portion of the device to improveits biocompatibility by reducing tissue reactions while also stimulatingangiogenesis and inhibiting biofouling. FIG. 1 illustrates one exampleof a glucose sensor (1—Teflon-covered Pt—Ir wire; 2—Ag/AgCl referencewire; 3—collagen scaffold; 4—electrically-insulating sealant; 5—Epoxy-Puouter membrane; 6—enzyme layer; 7—stripped and coiled Pt—Ir wire;8—cotton fiber with GOD gel). Examples of glucose sensors have beendescribed in published U.S. Patent Application No. 20070131549 and U.S.Pat. Nos. 6,475,750; 6,033,866; 6,965,791; and 6,893,545.

The three dimensional porous collagen scaffold can be provided in anysuitable manner. In some embodiments, the scaffold can be prepared byusing a freeze-drying method and reinforced using a nordihydroguaiareticacid (NDGA) solution.

The subject invention also concerns biosensors and other devicescomprising a biocompatible collagen scaffold. In particular embodiments,the device comprises a sensor, such as a glucose sensor. Sensors can be,but are not limited to electrochemical, optical, acoustic,piezoelectric, or thermoelectric sensors. In some embodiments, thecollagen scaffold is treated to form a polymer that intercalates intothe collagen scaffold, for example as is described in U.S. Pat. Nos.6,821,530 and 6,565,960. In some embodiments, the collagen scaffold istreated with a cross-linking compound comprising a reactive catechol ata pH sufficient to produce a reactive quinone. Typically, the pH used inthe reaction is neutral or alkaline. In one embodiment, the pH isbetween 7 to about 8. In another embodiment, the pH is between about 8to about 9, or about 9 to about 11. In a specific embodiment, thereactive catechol used for cross-linking is a di-catechol. In anexemplified embodiment, a collagen scaffold is embedded in an NDGAbisquinone polymer matrix. The device can also comprise an epoxy orother protective material layer. In some embodiments, the epoxy layer isunder the collagen scaffold. The device can also comprise anelectrically insulating layer. In some embodiments, the electricallyinsulating layer is under the collagen scaffold. Collagen scaffolds ofthe present invention can comprise open pores from about 10 μm to about200 μm in diameter, or from about 20 μm to 100 μm in diameter. In someembodiments, the collagen scaffold of the invention has a mean pore sizeof about 60 μm or less in diameter. In some embodiments, the collagenscaffold has a mean pore size of between about 40 μm and about 80 μm. Asnoted above, the collagen scaffolds can be loaded with varioustherapeutic compounds, such as compounds that have antimicrobialactivity, and/or that modulate inflammatory responses, angiogenesis,etc. In some embodiments, the collagen scaffold is loaded withantimicrobial, anti-inflammatory, and/or angiogenic compounds, drugs orgrowth factors.

Embodiments of the subject invention also concern methods for preparinga device for implantation into the body or tissue or animal wherein abiocompatible collagen scaffold of the present invention is preparedaround the exterior of at least a portion of the device. Optionally, thedevice or a portion thereof is coated with an epoxy layer, such asepoxy-polyurethane and/or an electrically insulating layer. In someembodiments, the method comprises contacting the device with a collagencontaining solution, followed by drying the collagen solution on thedevice, and then cross-linking and/or embedding the collagen in apolymerized matrix. In some embodiments, the collagen containingsolution comprises between about 0.5% to about 10% (w/v) collagen, andtypically about 1% (w/v) of collagen. In some embodiments, the collagencontaining solution is prepared in an acidic solution. The drying stepscan be by freeze-drying. The steps of contacting the device with acollagen solution, then drying the collagen solution can be repeatedmultiple times. In one embodiment, the steps are repeated about 2 to 4times. In a specific embodiment, the collagen can be embedded in apolymer matrix using a reactive catechol at a pH sufficient to produce areactive quinone that can polymerize to form a polymer. In someembodiments, the reaction solution for the reactive catechol is treatedso as to increase the levels of dissolved oxygen in the solution, suchas by sparging the solution with oxygen. Typically, the pH used in thereaction is neutral or alkaline. In one embodiment, the pH is between 7to about 8. In another embodiment, the pH is between about 8 to about 9,or about 9 to about 11. In an exemplified embodiment, the reactivecatechol is a di-catechol, such as NDGA. The collagen coated device canbe exposed to a solution of NDGA at a neutral or alkaline pH for asuitable period of time (e.g., 24 hours). The collagen scaffold isthereby embedded in an NDGA bisquinone polymer matrix. The scaffold canbe optionally washed following NDGA treatment. In some embodiments,after the collagen is treated to embed the collagen in polymerizedmatrix, the embedded collagen is subsequently freeze-dried. Optionally,the method can include loading a collagen scaffold with a compound,drug, growth factor, etc. that has antimicrobial, anti-inflammatory,and/or angiogenic activity. A flow chart of some embodiments of thesubject methods is shown in FIG. 21.

In some embodiments, the device is a biosensor having for example, anucleic acid, a protein, an organic compound or other molecule attached,coupled, or cross-linked thereto (for example, an enzyme can be attachedto an electrode of the sensor). Sensors can be used to detect glucose,alcohols, amino acids, drugs (and metabolites thereof), urea, hormones,and other compounds and analytes of interest. Sensors contemplated bythe present invention include, but are not limited to, sensors that candetect microorganisms, proteins, nucleic acids, organic compounds, suchas sugars and fatty acids, and other molecules or analytes.

As shown in FIG. 1, in an exemplified embodiment, the glucose sensor isa coil-type glucose sensor comprising a cross-linked enzyme, such asglucose oxidase. In one embodiment, the scaffold on the exterior of thedevice comprises open pores of about 10 μm to about 200 μm in diameter(mean), or from about 20 μm to about 100 μm in diameter. In a specificembodiment, the mean pore size of the collagen scaffold around theexterior of the device has a mean pore size of about 60 μm or less indiameter. In one embodiment, the mean pore size of the collagen scaffoldis between about 40 μm and about 80 μm in diameter. The sensor can alsooptionally comprise layers, such as an epoxy layer, and an electricallyinsulating layer. Collagen scaffolds of the invention can be loaded withvarious compounds that modulate inflammatory responses, angiogenesis,etc. In one embodiment, a collagen scaffold is loaded withantimicrobial, anti-inflammatory, and/or angiogenic drugs or growthfactors.

The subject invention also concerns methods for monitoring biologicalprocesses in vivo using a device of the invention that comprises abiocompatible collagen scaffold of the invention. In some embodiments,the device is a glucose sensor that can be used to monitor blood sugarlevels in a patient, such as a diabetic patient. In another embodiment,the device is a sensor that can detect hormone levels in a person oranimal, such as hormones associated with pregnancy. In otherembodiments, the device is a cardiac or nervous system monitor formonitoring heart, brain, etc. functions. In an exemplified embodiment, adevice of the invention is implanted into the body or tissue of a personor animal and the biological process that the device is capable ofmonitoring or detecting, etc. is monitored or detected. Biologicalprocesses can be monitored using the subject device for weeks, months,or years with the scaffold or coating providing resistance to biofoulingthereby promoting operational longevity in vivo.

The subject invention also concerns the use of collagen scaffolds of theinvention for the in vitro or in vivo delivery of bioactive compounds,drugs, growth factors, proteins, peptides, nucleic acids, inorganic ororganic molecules, etc. A collagen scaffold of the invention can beloaded with a bioactive compound, etc. and then the loaded scaffold canbe implanted or contacted with the body, tissue, cells, etc. of a personor animal. The compounds are then permitted to be released from thescaffold into the body, tissue, cell, etc. The collagen scaffold can beprovided on a biodegradable or non-degradable support structure ormatrix.

The collagen used in the present invention can be synthetic or derivedfrom any suitable animal species. The collagen can be from a vertebrateanimal or an invertebrate (e.g., starfish, sea urchin, sponges, etc.).In some embodiments, the collagen is fish, shark, skate, or raycollagen. In another embodiment, the collagen is human, equine, bovine,ovine, porcine, canine, or feline collagen. In an exemplifiedembodiment, the collagen is bovine collagen.

Collagen scaffolds of the present invention are stable both in vitro andin vivo for at least 4 weeks at body temperature. Also, scaffoldapplication around glucose sensors did not significantly affect thesensor's sensitivity. Scaffolds of the present invention can be used todeliver anti-inflammatory drugs and angiogenic growth factors (e.g.,VEGF, PDGF) in order to create a controlled local tissue environmentaround sensors with minimum inflammation and fibrosis but with increasedblood vessel density.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Materials and Methods

Materials

Type I collagen (purified from fetal bovine tendon) was a generous giftfrom Shriners Hospital for Children (Tampa, Fla.). Nordihydroguaiareticacid (NDGA) was purchased from Cayman Chemical Co. (Ann Arbor, Mich.).Glucose, bovine serum albumin (BSA) and 50% (w/w) glutaraldehyde (GA)were obtained from Fisher Scientific (Pittsburgh, Pa.). Glucose oxidase(GOD) (EC 1.1.3.4, Type X-S, Aspergillus niger, 157,500 U/g), epoxyadhesive (ATACS 5104), polyurethane (PU), tetrahydrofuran (THF) andcollagenase (EC 3.4.24.3, Type I, from Clostridium histolyticum, 302U/mg) were obtained from Sigma-Aldrich (St. Louis, Mo.). Sprague-Dawleyout-bred rats (male, 375-399 g) were purchased from Harlan (Dublin,Va.).

Preparation and Cross-Linking of Collagen Scaffold

The collagen scaffolds were prepared by a freeze-drying method. Collagenwas dissolved in 3% acetic acid to prepare a 1% (w/v) solution. Thesolution was applied to a cylinder-shaped polypropylene mold (Φ 10 mm,height 8 mm) and then freeze-dried. A cylindrical three dimensionalporous scaffold was obtained. The scaffolds were then cross-linked withNDGA or GA to minimize solubility and improve resistance to collagenasedegradation.

For NDGA cross-linking, dried collagen scaffolds were briefly soaked inabsolute ethanol, followed by soaking in 2 M of NaCl solution for 12 hat room temperature. Scaffolds were re-suspended in oxygen spargedphosphate buffered saline (PBS, 0.1 M NaH₂PO₄, pH 9.0) for 30 min atroom temperature. Scaffolds were then treated with 3 mg of NDGA in 1 mLof PBS as follow: NDGA was dissolved in 0.4 N NaOH at a concentration of30 mg/mL. One milliliter of the NDGA solution was added directly to PBSin which the scaffolds were suspended to a final concentration of 3mg/mL. The scaffolds were agitated in the NDGA solution for 24 h at roomtemperature. The scaffolds were removed, briefly rinsed with water andfreeze-dried.

For a comparative study of the effectiveness of the NDGA treatment,other scaffolds were treated with 0.5% GA for 2 h or 12 h in ethanolsolution at room temperature. To prevent the dissolution or loss of thematrix during the GA cross-linking process, 100% ethanol was usedinstead of water. The cross-linked scaffolds were washed with de-ionizedwater and freeze-dried again. The morphology of the scaffoldsbefore/after cross-linking was examined using scanning electronmicroscopy (SEM) after gold sputter coating of the samples in a metalevaporator according to standard procedures.

To evaluate the stability of the scaffold after cross-linking, thedegree of cross-linking (Dc) was estimated by weighing the dried samplesbefore and after cross-linking. Dc was calculated using the followingequation:Dc[%]=(sample mass after cross-linking/sample mass beforecross-linking)×100

The swelling property of the porous scaffolds was examined by measuringwater absorption. The scaffolds were weighed after thorough drying(W_(dry)) and immersed in purified water. After 24 h, the scaffolds wereremoved from the water and immediately weighed again (W_(wet)). Waterabsorption was calculated by using the following equation:Water absorption(%)=[(W _(wet) −W _(dry))/W _(wet)]×100In Vitro and In Vivo Evaluation of the Collagen Scaffolds

In order to examine the biological stability of the cross-linkedscaffolds, in vitro and in vivo biodegradation tests were performed. Invitro biodegradation of NDGA and GA cross-linked scaffolds was testedusing bacterial collagenase. Fabricated NDGA and GA cross-linkedcollagen scaffolds were incubated in the collagenase solution (1 mg/mLin PBS at 37° C.) for up to 4 weeks. Scaffolds were removed from thesolution, rinsed with de-ionized water and freeze-dried at given timeintervals (weeks 1 to 4) during incubation. The in vitro degradation wasevaluated as the percentage of weight difference of the dried scaffoldbefore and after enzyme digestion.

In order to determine the stability of the cross-linked scaffolds invivo, NDGA and GA cross-linked collagen scaffolds were directlyimplanted in rats. The scaffolds were disinfected with 70% ethanolsolution for 2 h and implanted subcutaneously in the back of the rats.Scaffolds were explanted at 7, 14, 21, and 28 days after implantation.After explantation, the scaffolds were examined macroscopically.

Preparation of Porous Collagen Scaffolds Around Implantable GlucoseSensors

Coil-type glucose sensors loaded with cross-linked enzyme (GOD: GlucoseOxidase) were fabricated using a Platinum-Iridium (Pt/Ir) wire (Tefloncoated, Φ 0.125 mm, Pt:Ir=9:1, Medwire, Sigmund Cohn Corp., MountVernon, N.Y.). Then, bovine tendon type I collagen scaffolds wereapplied around the sensors (FIG. 1). Briefly, in order to fabricate aglucose sensor, the Teflon coating of the top 10 mm of a Pt/Ir wire wasremoved and the wire was wound up along a 30-gauge needle to form acoil-like cylinder. The cylinder unit had an outer diameter of 0.55 mmand an inner diameter of 0.3 mm and a length of 1 mm. A cotton threadwas inserted inside the coil chamber to retain the enzyme solutionduring enzyme coating of the electrodes. GOD was added and cross-linkedto the sensors by dip coating in an aqueous solution containing 1% GOD,4% BSA, and 0.6% (w/w) glutaraldehyde. The outer membrane of the sensorwas coated with Epoxy-Polyurethane (Epoxy-PU) by dipping in Epoxy-PUsolution (2.5% (w/v) in THF, Epoxy:PU=1:1). The sensor was dried at roomtemperature for at least 24 h. The two ends of the sensing element weresealed by electrically-insulating sealant (Brush-On electrical tape,North American Oil Company) (Long et al., 2005; Yu et al., 2006).

To apply collagen scaffolds around the sensors, the sensors weredip-coated with 1% (w/v) collagen solution and freeze-dried. The porousscaffolds around the glucose sensors were cross-linked with either NDGAor GA as previously described. Obtained sensors were stored dry at roomtemperature or in PBS at 4° C. The morphology of the sensors wasobserved using light microscope and SEM.

Silver wires (Teflon coated, Φ 0.125 mm, World Precision Instruments,Inc.) were used to fabricate the Ag/AgCl reference electrodes. Silverwires were coiled and anodized galvanostatically at 1 mA overnight instirred 0.1 M HCl (Long et al., 2005; Yu et al., 2006).

In Vitro Characterization of Sensors Coated with Scaffolds

The glucose sensors were characterized in PBS (pH 7.4) at 700 mV versusthe incorporated Ag/AgCl reference electrodes. The working electrode(Pt/Ir wire) and Ag/AgCl reference electrode of each sensor wereconnected to an Apollo 4000 potentiostat (World Precision Instruments,Inc., Sarasota, Fla.). The background current was allowed to stabilizefor 10 minutes, and the sensors were then exposed to a series of glucosesolutions in order to examine their sensitivities and linearities. Theresponse sensitivity (S) was repeatedly assessed by 1) measuring theresponse current (I₁) of a C₁ glucose solution, 2) adding a concentratedglucose solution into the measured solution to increase the glucoseconcentration to C₂ and 3) measuring the response current (I₂) of theresulting solution. The sensitivity was expressed as the currentincrease caused by a 1 mM glucose increase, i.e. S=(I₂−I₁)/(C₂−C₁).

In Vivo Anti-Inflammation Effect of the NDGA-Crosslinked Scaffold

To evaluate in vivo anti-inflammatory effect of the NDGA-crosslinking,NDGA and GA- (control) crosslinked scaffolds were implantedsubcutaneously in the back of the Sparague-Dawley rats. The subcutaneoustissue samples around the implanted site were explanted 3-, 7-, 14-,21-, 28-, and 49-days post implantation. At set time intervals, tissuesamples were collected and fixed in situ (10% buffered formalin). Thefixed tissue samples were embedded in paraffin and sectioned with 10 μmthickness. Various sections were stained with hematoxylin and eosin(H&E) and imaged using microscope with digital camera.

FIG. 4 shows the inflammation response associated with implanting abiosensor according to the invention.

EXAMPLE 1 Preparation of Porous Cross-Linked Collagen Scaffolds

The chemistry of the NDGA cross-linking reaction differs from thereaction using the GA treatment (FIG. 2). GA is the most commoncross-linking agent used for fixation of collagen scaffolds for tissuebioengineering. Both aldehyde functional groups of the GA molecule reactwith amine groups between two neighboring polypeptide chains,particularly lysine side chains. Unfortunately, GA cross-linking isencumbered with potential cytotoxicity problems caused by the presenceof unreacted residual groups and/or the release of monomers and smallpolymers during enzymatic degradation (Huang-Lee et al., 1990; van Luynet al., 1992).

NDGA is an alternative cross-linking agent which possesses reactivecatechols. Collagen cross-linking with NDGA mimics the quinine tanningmechanism in the skate egg capsule. Catechol-quinone tanning systems areprevalent in a wide variety of animals, which the process serves tostrengthen vulnerable extracellular matrices (e.g. insect cuticle,mussel byssus threads) (Koob et al., 2002a; Koob et al., 2004). NDGA,isolated from the creosote bush, is a low molecular weight di-catecholcontaining two ortho-catechols. The two catechols on NDGA undergoauto-oxidation at neutral or alkaline pH producing reactive quinones.Two quinones then couple via aryloxy free radical formation andoxidative coupling, forming bisquinone crosslinks at each end. The NDGAcontinues forming a large cross-linked bisquinone polymer network inwhich the collagen fibrils are embedded. The NDGA treatment can alsoform crosslinks with amino acid side chains of collagen (Koob et al.,2002a; Koob et al., 2002b; Koob et al., 2004).

In this study, highly porous collagen scaffolds were prepared by afreeze-drying method. It was ascertained that the scaffolds prepared asdescribed herein have an open cell and interconnected pore structurebased on SEM observation (FIG. 3A). The pores of the scaffolds areregularly distributed and range from 20 to 100 μm in diameter (mean ˜60em). Sharkawy et al., 1997 reported that the a 60 μm mean-pore-sizedpolyvinyl alcohol (PVA) sponge provided a tissue in-growth environmentand allows for infiltration of neovasculature but did not allow forfibrous tissue in-growth. After cross-linking with NDGA and GA, the poresize and pore structure of both scaffolds are not significantly altered(FIG. 3B and FIG. 3C). FIG. 4 shows the degree of cross-linking andwater absorption of the scaffolds using different cross-linking methods.The mass was reduced to about 70% after NDGA treatment and 60% with GAtreatment after the cross-linking process due to the loss ofuncross-linked collagen components. Cross-linked collagen scaffolds hada significantly higher form stability than uncross-linked collagenscaffolds. Also, the swelling behavior of both NDGA and GA cross-linkedscaffolds showed no significant differences between the two differentcross-linking agents. The water absorptions of both cross-linkedscaffolds were above 99%. The high swelling property of sponge-likematrices seems to be dependent on the porous inner structure of thescaffold, which possesses good absorbent characteristics (Patel et al.,1996).

EXAMPLE 2 In Vitro and In Vivo Evaluation of Porous Collagen Scaffolds

The biological stability of the cross-linked collagen scaffolds wasinvestigated by in vitro and in vivo biodegradation tests. Degradationin both uncross-linked (control) and cross-linked scaffolds wascharacterized by determining weight loss of the scaffold after enzymaticdigestion. The uncross-linked scaffolds and scaffolds cross-linked withGA for 2 hours were completely degraded in the collagenase solutionwithin several hours while NDGA or GA cross-linked (for 12 h) scaffoldswere not degraded within 24 hours. A significant increase in resistanceto enzymatic digestion could be shown after cross-linking. FIG. 5 showslong-term collagenase in vitro degradation test (weight remaining %) ofthe NDGA and GA cross-linked scaffolds. After one week exposure tocollagenase, both types of scaffolds showed high resistance to enzymaticdigestion (>80% weight remaining). After three and four weeks, allscaffolds retained 70% of their initial mass. However, in the case of GAcross-linked scaffold, the pore size was increased after 4 weekscollagenase digestion process (FIG. 6B vs FIG. 6D). In contrast, thepore size of NDG scaffolds did not appear to increase (FIG. 6A and FIG.6C). This result suggests that NDGA or GA treatment can provide collagenscaffolds with improved enzymatic biodegradation stability. Thecollagenase cleavage sites were more effectively blocked by thecross-linking of the collagen scaffolds (Angele et al., 2004).

In order to study the stability of the cross-linked scaffolds in vivo,cross-linked collagen scaffolds were implanted in the subcutaneoustissue of the Sprague-Dawley rats and explanted samples two and fourweeks post implantation. After two weeks implantation, the NDGAcross-linked scaffolds did not show evidence of physical damage, but theoverall shape of the GA cross-linked scaffolds was deformed and the sizeslightly reduced (FIG. 7A). After four weeks, the size and shape of theGA cross-linked scaffolds were dramatically changed but there was nosignificant change in NDGA cross-linked scaffolds (FIG. 7B). Thisindicated that NDGA treatment can much more improve the physicalstability of the scaffolds in vivo than GA treatment, presumably becauseof greater mechanical properties of NDGA cross-linked collagen. Koob etal., 2002b tested NDGA cross-linked collagen fibers and reported thatthe ultimate tensile strength of NDGA cross-linked fibers weresignificantly greater than those of GA cross-linked fibers because NDGAdose not chemically crosslink the collagen unlike GA cross-linking.Instead, the collagen fibrils are embedded in a polymerized NDGA matrix,i.e., a fiber reinforced composite (FIG. 2).

EXAMPLE 3 Porous Collagen Scaffolds Around Implantable Glucose Sensors

Coil-type glucose sensors loaded with cross-linked enzyme (GOD: GlucoseOxidase) were first fabricated by using Platinum-Iridium (Pt/Ir) wires.Then, bovine tendon type I collagen scaffolds were applied around thesensors (FIG. 1). Yu et al., 2006 previously reported that this“coil-type” sensor allows more GOD loading, provides a largerelectrochemical surface area, and therefore increases the responsecurrent as compared to a “needle-type” sensor. The coil-type sensor ofthe invention is flexible and miniaturized (0.5 mm dia.) forsubcutaneous implantation. It is composed of a two-electrode system witha glucose indicating platinum electrode and a Ag/AgCl reference-counterelectrode. A sensor of the present invention utilizes a three-layermembrane configuration of cross-linked collagen scaffold,epoxy-polyurethane (Epoxy-PU) and GOD. The collagen scaffold (the outerlayer in this case) can uptake 99% of its dry weight of water includingglucose and other molecules. The Epoxy-PU membrane under the scaffold ispermeable to glucose and oxygen but impermeable to most interferingsubstances. GOD immobilized in a BSA/GA matrix is sandwiched between thePt/Ir wire and the Epoxy-PU membrane. In order to eliminate air bubblesentrapped in the chamber during coating, to stabilize the enzyme gelinside the chamber, and to make the enzyme solution easier to remain inthe coil, cotton fiber was used inside the coil chamber. The collagenscaffolds were prepared by a freeze-drying method and cross-linked tominimize water solubility and enzymatic collagenase degradation. A lightmicroscope, was used to confirm that the porous scaffolds thoroughlysurrounded the sensor tips (FIG. 8A and FIG. 8B). The surface andcross-sectional morphology of the scaffolds around the sensors were alsoobserved using SEM. Many collagen fibrils and uniform open porestructure were observed on the surface (FIG. 8C). Inter-connected openpores in the scaffold and a thickness of 150-200 μm were observed incross-sectional region (FIG. 8D).

The amperometric response curves of the glucose sensors with and withoutscaffold (control) were obtained by varying the glucose concentrationfrom 5 mM to 15 mM as shown in FIG. 9. These glucose concentrations wereselected because these concentrations were located in the linearresponse region (2-30 mM) of the studied sensors. The results showed nosignificant response current change before and after scaffoldapplication around the sensor. However, the sensors with scaffolds had aslower response time to reach equilibrium current (T_(95%)) than controlsensors. The response time, T_(95%), is defined as the time at 95% ofthe maximum current change (I₂−I₁). The T_(95%) of control sensor was14.0 min whereas T_(95%) of the sensors with NDGA- and GA cross-linkedscaffold were 17.9 min and 17.0 min, respectively. The delay of theresponse time (17.9 and 17 min) was probably caused by the addedphysical barrier of the porous scaffolds.

The currents produced by sensors with NDGA- and GA cross-linkedscaffolds and by sensors without scaffolds in response to varyingglucose concentration (2-30 mM) are shown in FIG. 10. The responsecurrents of the control sensors in the high glucose concentration region(20-30 mM) were only a little higher than those of the sensors havingscaffolds. The average sensitivity of the control, NDGA- and GAcross-linked scaffold around sensors was 11.0, 7.1, and 8.1 nA/mM,respectively. Therefore, scaffold application around the glucose sensorsdid not negatively affect the function of the sensors.

The sensitivity changes of the sensors with varying wall thickness ofthe scaffold controlled by dipping cycles in collagen solution wereexamined. As can be seen in FIG. 11, the sensitivity of sensorsdip-coated four times remained at 60% of their initial sensitivity(i.e., without a scaffold layer). When the sensors were dip-coated morethan 5 times, glucose could not diffuse properly through the scaffoldsand the sensitivity was reduced to below 20% of the initial sensitivity.Although the porous scaffold material has good water absorbentproperties, the wall thickness can affect the sensor function.

EXAMPLE 4 In Vitro and In Vivo Evaluation of Implantable Glucose Sensor

An implantable glucose sensor used in these studies is shown in FIG. 13having a multi-layered sensing element as shown in FIG. 1. The woundclip is shown as 10, the Ag/AgCl Reference Counter Electrode is shown as20, the loop is shown as 30, and the sensor with scaffold Pt/Irelectrode is labeled 40. In the in vitro study, 16 sensors were placedin PBS (pH 7.4) at 37° C. for four weeks. Sensitivity was measured atgiven time intervals (weeks 1 to 4) using potentiostat (WPI, Inc.).Sensitivity (nA/mM)=(I_(15 mM)−I_(5 mM))/(15 mM−5 mM). In the in vivostudy, 48 sensors (24-short wire) were implanted subcutanteously in thebacks of rats and sensitivities measured weekly under anesthesia using a4-channel potentiostat (WPI, Inc.) (see FIGS. 14 and 15). Sensitivity(nA/mM)=(I_(max)−I₀)/(C_(max)−C₀). Concentration of blood glucose (C)was measured using a standard FREESTYLE Glucometer.

As shown in FIG. 16, hydrated NDGA-reinforced collagen scaffolds hadhigher form stability than GA cross-linked collagen scaffolds. In regardto in vitro sensitivity, a slight decrease of the sensitivity wasobserved in the presence of the scaffold. However, both sensors withscaffold retained above 80% of their original sensitivity up to fourweeks in vitro (see FIG. 17). Thus, three dimensional scaffoldapplication around glucose sensors did not seriously affect sensorsensitivity in vitro.

In regard to in vivo sensitivity of the implanted glucose sensor,short-wired sensors showed better performance overall than long-wiredsensors due to limitation of micro-motion in short-wired sensors.Sensors with NDGA-reinforced scaffolds retained much higher sensitivityafter four weeks implantation than sensors with GA cross-linkedscaffolds (see FIG. 18).

In regard to physical stability and sensitivity of the implanted glucosesensor, short-wired sensors with NDGA-reinforced collagen scaffolds hadmuch higher sensitivity and physical stability than sensors with AGcross-linked scaffolds during four weeks in vivo (see FIGS. 19 and 20).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

REFERENCES

-   U.S. Pat. No. 6,821,530-   U.S. Pat. No. 6,565,960-   Angele P, Abke J, Kujat R, Faltermeier H, Schumann D, Nerlich M,    Kinner B, Englert C, Ruszczak Z, Mehrl R and others. Influence of    different collagen species on physico-chemical properties of    crosslinked collagen matrices. Biomaterials 2004; 25(14):2831-41.-   Armour J C, Lucisano J Y, McKean B D, Gough D A. Application of    chronic intravascular blood glucose sensor in dogs. Diabetes 1990;    39(12):1519-26.-   Ash S R, Poulos J T, Rainier J B, Zopp W E, Janle E, Kissinger P T.    Subcutaneous capillary filtrate collector for measurement of blood    glucose. Asaio J 1992; 38(3):M416-20.-   Barbani N, Giusti P, Lazzeri L, Polacco G, Pizzirani G.    Bioartificial materials based on collagen: 1. Collagen cross-linking    with gaseous glutaraldehyde. J Biomater Sci Polym Ed 1995;    7(6):461-9.-   Bindra D S, Zhang Y, Wilson G S, Sternberg R, Thevenot D R, Moatti    D, Reach G. Design and in vitro studies of a needle-type glucose    sensor for subcutaneous monitoring. Anal Chem 1991; 63(17):1692-6.-   Chvapil M, Kronenthal L, Van Winkle W, Jr. Medical and surgical    applications of collagen. Int Rev Connect Tissue Res 1973; 6:1-61.-   Ertefai S, Gough D A. Physiological preparation for studying the    response of subcutaneously implanted glucose and oxygen sensors. J    Biomed Eng 1989; 11(5):362-8.-   Frost M C, Meyerhoff M E. Implantable chemical sensors for real-time    clinical monitoring: progress and challenges. Curr Opin Chem Biol    2002; 6(5):633-41.-   Huang-Lee L L, Cheung D T, Nimni M E. Biochemical changes and    cytotoxicity associated with the degradation of polymeric    glutaraldehyde derived crosslinks. J Biomed Mater Res 1990;    24(9):1185-201.-   Johnson K W, Mastrototaro J J, Howey D C, Brunelle R, Burden-Brady P    L, Bryan N A, Andrew C C, Rowe H M, Allen D J, Noffke B W and    others. In vivo evaluation of an electroenzymatic glucose sensor    implanted in subcutaneous tissue. Biosens Bioelectron 1992;    7(10):709-14.-   Joseph J I, Torjman M J. Glucose Sensors. In: Wnek G E, Bowlin G L,    editors. Encyclopedia of biomaterials and biomedical engineering.    New York: Marcel Dekker; 2004. p 683-692.-   Kerner W, Kiwit M, Linke B, Keck F S, Zier H, Pfeiffer E F. The    function of a hydrogen peroxide-detecting electroenzymatic glucose    electrode is markedly impaired in human sub-cutaneous tissue and    plasma. Biosens Bioelectron 1993; 8(9-10):473-82.-   Khor E. Methods for the treatment of collagenous tissues for    bioprostheses. Biomaterials 1997; 18(2):95-105.-   Koob T J, Hernandez D J. Material properties of polymerized    NDGA-collagen composite fibers: development of biologically based    tendon constructs. Biomaterials 2002b; 23(1):203-12.-   Koob T J, Willis T A, Hernandez D J. Biocompatibility of    NDGA-polymerized collagen fibers. I. Evaluation of cytotoxicity with    tendon fibroblasts in vitro. J Biomed Mater Res 2001a; 56(1):31-9.-   Koob T J, Willis T A, Qiu Y S, Hernandez D J. Biocompatibility of    NDGA-polymerized collagen fibers. II. Attachment, proliferation, and    migration of tendon fibroblasts in vitro. J Biomed Mater Res 2001b;    56(1):40-8.-   Koob T J. Biomimetic approaches to tendon repair. Comp Biochem    Physiol A Mol Integr Physiol 2002a; 133(4):1171-92.-   Koob T J. Collagen Fixation. In: Wnek G E, Bowlin G L, editors.    Encyclopedia of biomaterials and biomedical engineering. New York:    Marcel Dekker; 2004. p 335-347.-   Koudelka M, Rohner-Jeanrenaud F, Terrettaz J, Bobbioni-Harsch E, de    Rooij N F, Jeanrenaud B. In-vivo behaviour of hypodermically    implanted microfabricated glucose sensors. Biosens Bioelectron 1991;    6(1):31-6.-   Lee C H, Singla A, Lee Y. Biomedical applications of collagen. Int J    Pharm 2001; 221(1-2):1-22.-   Lee S, Nayak V, Dodds J, Pishko M, Smith N B. Glucose measurements    with sensors and ultrasound. Ultrasound Med Biol 2005; 31(7):971-7.-   Long N, Yu B, Moussy Y, Moussy F. Strategies for testing long-term    transcutaneous amperometric glucose sensors. Diabetes Technol Ther    2005; 7(6):927-36.-   Meyerhoff C, Bischof F, Sternberg F, Zier H, Pfeiffer E F. On line    continuous monitoring of subcutaneous tissue glucose in men by    combining portable glucosensor with microdialysis. Diabetologia    1992; 35(11):1087-92.-   Moscone D, Mascini M. Microdialysis and glucose biosensor for in    vivo monitoring. Ann Biol Clin (Paris) 1992; 50(5):323-7.-   Moussy F, Harrison D J, O'Brien D W, Rajotte R. Performance of    subcutaneously implanted needle-type glucose sensors employing a    novel trilayer coating. Anal Chem 1993; 65(15):2072-7.-   Moussy F, Harrison D J, Rajotte R A miniaturized Nafion-based    glucose sensor: in vitro and in vivo evaluation in dogs. Int J Artif    Organs 1994b; 17(2):88-94.-   Moussy F, Harrison D J. Prevention of the rapid degradation of    subcutaneously implanted Ag/AgCl reference electrodes using polymer    coatings. Anal Chem 1994c; 66(5):674-9.-   Moussy F, Jakeway S, Harrison D J, Rajotte R. In vitro and in vivo    performance and lifetime of perfluorinated ionomer-coated glucose    sensors after high-temperature curing. Anal Chem 1994a;    66(22):3882-8.-   Pachence J M. Collagen-based devices for soft tissue repair. J    Biomed Mater Res 1996; 33(1):35-40.-   Patel V R, Amiji M M. Preparation and characterization of    freeze-dried chitosan-poly(ethylene oxide) hydrogels for    site-specific antibiotic delivery in the stomach. Pharm Res 1996;    13(4):588-93.-   Pickup J C, Shaw G W, Claremont D J. In vivo molecular sensing in    diabetes mellitus: an implantable glucose sensor with direct    electron transfer. Diabetologia 1989; 32(3):213-7.-   Pieper J S, van der Kraan P M, Hafmans T, Kamp J, Buma P, van    Susante J L, van den Berg W B, Veerkamp J H, van Kuppevelt T H.    Crosslinked type II collagen matrices: preparation,    characterization, and potential for cartilage engineering.    Biomaterials 2002; 23(15):3183-92.-   Quinn C P, Pathak C P, Heller A, Hubbell J A. Photo-crosslinked    copolymers of 2-hydroxyethyl methacrylate, poly(ethylene glycol)    tetra-acrylate and ethylene dimethacrylate for improving    biocompatibility of biosensors. Biomaterials 1995; 16(5):389-96.-   Reddy S M, Vadgama P M. Ion exchanger modified PVC    membranes—selectivity studies and response amplification of oxalate    and lactate enzyme electrodes. Biosens Bioelectron 1997;    12(9-10):1003-12.-   Reichert W M, Saavedra S S. Materials considerations in the    selection, performance, and adhesion of polymeric encapsulants for    implantable sensors. In: Williams D F, editor. Medical and Dental    Materials. New York: VCH Publishers, Inc.; 1992. p 303-343.-   Reichert W M, Sharkawy A A. Chap. 28 Biosonsors In: Von Recum A,    editor. Handbook of biomaterials evaluation: scientific, technical,    and clinical testing of implant materials. Philadelphia: Taylor &    Francis; 1999. p 439-460.-   Rigby G P, Crump P, Vadgama P. Open flow microperfusion: approach to    in vivo glucose monitoring. Med Biol Eng Comput 1995; 33(2):231-4.-   Sharkawy A A, Klitzman B, Truskey G A, Reichert W M. Engineering the    tissue which encapsulates subcutaneous implants. I. Diffusion    properties. J Biomed Mater Res 1997; 37(3):401-12.-   Sharkawy A A, Neuman M R, Reichert W M. Sensocompatibility: Design    considerations for biosensor-based drug delivery systems. In: Park    K, editor. Controlled Drug Delivery: The Next Generation.    Washington, D.C.: American Chemical Society, 2007.-   Shaw G W, Claremont D J, Pickup J C. In vitro testing of a simply    constructed, highly stable glucose sensor suitable for implantation    in diabetic patients. Biosens Bioelectron 1991; 6(5):401-6.-   Sheu M T, Huang J C, Yeh G C, Ho H O. Characterization of collagen    gel solutions and collagen matrices for cell culture. Biomaterials    2001; 22(13):1713-9.-   Shichiri M, Asakawa N, Yamasaki Y, Kawamori R, Abe H. Telemetry    glucose monitoring device with needle-type glucose sensor: a useful    tool for blood glucose monitoring in diabetic individuals. Diabetes    Care. Volume 9; 1986. p 298-301.-   Shichiri M, Yamasaki Y, Nao K, Sekiya M, Ueda N. In vivo    characteristics of needle-type glucose sensor—measurements of    subcutaneous glucose concentrations in human volunteers. Horm Metab    Res Suppl 1988; 20:17-20.-   Sung H W, Hsu H L, Shih C C, Lin D S. Cross-linking characteristics    of biological tissues fixed with monofunctional or multifunctional    epoxy compounds. Biomaterials 1996; 17(14):1405-10.-   van Luyn M J, van Wachem P B, Olde Damink L H, Dijkstra P J, Feijen    J, Nieuwenhuis P. Secondary cytotoxicity of cross-linked dermal    sheep collagens during repeated exposure to human fibroblasts.    Biomaterials 1992; 13(14):1017-24.-   Wilkins E, Atanasov P, Muggenburg B A. Integrated implantable device    for long-term glucose monitoring. Biosens Bioelectron 1995;    10(5):485-94.-   Yu B, Long N, Moussy Y, Moussy F. A long-term flexible    minimally-invasive implantable glucose biosensor based on an    epoxy-enhanced polyurethane membrane. Biosens Bioelectron 2006;    21(12):2275-82.

1. A biocompatible collagen scaffold and/or coating for use with adevice implantable in the body or tissue of a person or animal, whereinsaid collagen scaffold or coating is embedded within a polymer matrix.2. The collagen scaffold according to claim 1, wherein said collagenscaffold is treated with a cross-linking compound comprising a reactivecatechol at a pH sufficient to produce a reactive quinone.
 3. Thecollagen scaffold according to claim 2, wherein said reactive catecholis a di-catechol.
 4. The collagen scaffold according to claim 1, whereinsaid collagen scaffold is embedded in a nordihydroguaiaretic acid (NDGA)bisquinone polymer matrix.
 5. The collagen scaffold according to claim1, wherein said device comprises a sensor.
 6. The collagen scaffoldaccording to claim 5, wherein said sensor is a glucose sensor.
 7. Thecollagen scaffold according to claim 1, wherein said collagen scaffoldcomprises open pores of between about 10 μm to about 200 μm in diameter(mean).
 8. The collagen scaffold according to claim 7, wherein the meanpore size of said collagen scaffold is about 60 μm or less in diameter.9. The collagen scaffold according to claim 7, wherein the mean poresize of said collagen scaffold is between about 40 μm and about 80 μm indiameter.
 10. The collagen scaffold according to claim 1, wherein saidscaffold comprises at least one of an antimicrobial, anti-inflammatory,and/or angiogenic compound, drug or growth factor.
 11. A method forpreparing a device for implantation into the body or tissue of a personor animal, said method comprising placing a biocompatible collagenscaffold or coating on said device, wherein said collagen scaffold orcoating is embedded within a polymer matrix.
 12. The method according toclaim 11, wherein said method comprises: a) contacting said device witha collagen containing solution; b) drying said collagen solution on saiddevice; and c) embedding said collagen on said device in a polymermatrix.
 13. The method according to claim 12, wherein said collagen isembedded in said matrix using a reactive catechol at a pH sufficient toproduce a reactive quinone.
 14. The method according to claim 13,wherein said reactive catechol is a dicatechol.
 15. The method accordingto claim 14, wherein said dicatechol is NDGA.
 16. The method accordingto claim 12, wherein steps (a) and (b) of said method are repeated atleast one time.
 17. The method according to claim 12, wherein steps (a)and (b) of said method are repeated at least two to four times.
 18. Themethod according to claim 12, wherein said drying step comprisesfreeze-drying.
 19. The method according to claim 12, wherein saidcollagen embedded in said matrix is subsequently freeze-dried.
 20. Themethod according to claim 12, wherein said collagen containing solutionof step (a) comprises about 1% (w/v) collagen.
 21. The method accordingto claim 12, wherein said collagen scaffold comprises open pores ofabout 10 μm to about 200 μm in diameter (mean).
 22. The method accordingto claim 21, wherein the mean pore size of the collagen scaffold isabout 60 μm or less in diameter.
 23. The method according to claim 21,wherein the mean pore size of the collagen scaffold is between 40 μm and80 μm in diameter.
 24. A method for providing an implantable device witha biocompatible collagen coating or scaffold, wherein said collagenscaffold or coating is embedded within a polymer matrix, said methodcomprising: a) contacting an implantable device structure with acollagen containing solution; b) drying said collagen solution on saidstructure; and c) embedding said collagen of said structure in a polymermatrix.
 25. The method according to claim 24, wherein said collagen isembedded in said matrix using a reactive catechol at a pH sufficient toproduce a reactive quinone.
 26. The method according to claim 25,wherein said reactive catechol is a dicatechol.
 27. The method accordingto claim 26, wherein said dicatechol is NDGA.
 28. The method accordingto claim 24, wherein steps (a) and (b) of said method are repeated atleast one time.
 29. The method according to claim 24, wherein steps (a)and (b) of said method are repeated a plurality of times.
 30. The methodaccording to claim 24, wherein said drying step is freeze-drying. 31.The method according to claim 24, wherein said collagen embedded in saidmatrix is subsequently freeze-dried.
 32. The method according to claim24, wherein said collagen containing solution of step (a) comprisesabout 1% (w/v) collagen.
 33. The method according to claim 24, whereinsaid collagen scaffold comprises open pores of about 10 μm to about 200μm in diameter (mean).
 34. The method according to claim 33, wherein themean pore size of the collagen scaffold is about 60 μm or less indiameter.
 35. The method according to claim 33, wherein the mean poresize of the collagen scaffold is between 40 μm and 80 μm in diameter.36. A device having enhanced biocompatibility for implantation into thebody or tissue of a person or animal, wherein said device comprises abiocompatible collagen scaffold or coating embedded within a polymermatrix.
 37. The device according to claim 36, wherein said scaffoldand/or coating is treated with a cross-linking compound comprising areactive catechol at a pH sufficient to produce a reactive quinone. 38.The device according to claim 37, wherein said reactive catechol is adi-catechol.
 39. The device according to claim 36, wherein said collagenscaffold and/or coating is embedded in a nordihydroguaiaretic acid(NDGA) bisquinone polymer matrix.
 40. The device according to claim 36,wherein said device is a sensor.
 41. The device according to claim 36,wherein said device is a glucose sensor.
 42. The device according toclaim 36, wherein said device comprises an epoxy coating underneath saidcollagen scaffold and/or coating.
 43. The device according to claim 42,wherein said epoxy coating is an epoxy-polyurethane coating.
 44. Thedevice according to claim 36, wherein said scaffold comprises open poresof about 10 μm to about 200 μm in diameter (mean).
 45. The deviceaccording to claim 44, wherein the mean pore size of said collagenscaffold is about 60 μm or less in diameter.
 46. The device according toclaim 44, wherein the mean pore size of said collagen scaffold isbetween about 40 μm and about 80 μm in diameter.
 47. The deviceaccording to claim 36, wherein said scaffold comprises an antimicrobial,anti-inflammatory, and/or angiogenic compound, drug or growth factor.